(1) Field of the Invention
The present invention relates to a purified protein from Actinobacillus pleuropneumoniae (APP) outer membrane (OM) which is useful as a vaccine in swine. In particular, the present invention relates to a protein which has a molecular weight of about 43 Kd as measured by SDS PAGE, is not heat modifiable and is soluble in a 1% by weight to volume aqueous solution of sodium lauryl sarcosinate (SLS).
(2) Prior Art
Most prior art relevant to bacterial vaccines is based upon killing a virulent strain of the bacteria by using formalin or by heat killing the bacteria. Generally the bacterial cells are a single serotype of the target bacteria. As a result, heterologous serotypes of the same bacteria can cause disease in spite of vaccination. The reason for this is not completely understood; however, it appears that antibodies to the heterologous serotype are not induced by the vaccine and thus there is no protective immunity.
With APP, there are multiple serotypes of the infecting organism. The serotypes are characterized based on antigenic differences in the capsular polysaccharides. Infection with one serotype provides protection against subsequent challenge with all serotypes. However, formalinized or heat-killed bacterins provide moderate protection against the homologous serotype and essentially no protection against heterologous serotypes. Research on antigens of APP and the immune response of swine to those antigens showed that there are a variety of antigenically similar outer membrane proteins that are found in all isolates of APP, regardless of serotype, which contribute to cross-protective immunity.
Mulks, M., and Thacker, B. in Proc. Int. Pig Veter. Soc. 10 81 (1988) describe an OM vaccine for pigs derived from Haemophilus pleuropneumoniae (now known as Actinobacillus pleuropneumoniae or APP). The vaccine contained APP outer membranes. The OM was produced by sonication of lysozyme-sucrose treated cells followed by sucrose density gradient centrifugation. The sonication was for 10-15 seconds. Lysozyme degrades peptidoglycan (the cell wall). Sucrose maintains the cell membranes remaining after treatment with the lysozyme until the cells are sonicated. No preservative was used in the preparation of the vaccine. Sucrose density gradient centrifugation and separation of OM is not a commercially viable method for producing the vaccine.
Other references for work on APP vaccines are: (1) P. J. Fedorka-Cray, M. J. Huether, D. L. Stine, and G. A. Anderson. Efficacy of a cell extract form Actinobacilius (Haemophilus) pleuropneumoniae serotype 1 against disease in swine. Infect. Immun. 58:358-365 (1990). Two experimental APP vaccines were tested: (a) APP broth culture supernatant concentrated with 20% polyethylene glycol, containing primarily extracellular hemolysin; (b) OM prepared by a SLS extraction procedure. Both vaccines provided significant, although not complete, protection against homologous challenge as compared to unvaccinated controls.
(2) J. Devenish, S. Rosendal, and J. T. Bosse. Humoral antibody response and protective immunity in swine following immunization with the 104 kilodalton hemolysin of Actinobacillus pleuropneumoniae. Infect. Immun. 58:3829-3832 (1990). Purified 104 Kd hemolysin was tested as a vaccine, and elicited complete protection against mortality and significant, although far from complete, protection against lung involvement.
(3) D. K. Lenser, T. L. McDonald, and N. G. Miller. Protection of mice against the lethal effect of an intraperitoneal infection with Haemophilus (Actinobacillus) pleuropneumoniae after vaccination with capsular proteins. Veter. Microbiol. 18:335-348 (1988). The vaccines tested were: (a) whole cell bacterin; (b) capsular polysaccharide; (c) outer membranes prepared by a SLS extraction procedure; and (d) lipopolysaccharide (LPS) vaccines against APP in a mouse model. The results were: (a) whole cell bacterin gave some homologous but no heterologous protection; (b) capsular polysaccharide vaccine gave good homologous and no heterologous protection; (c) OM vaccine provided limited homologous and no heterologous protection; and (d) LPS vaccine provided no protection.
European Patent Application No. 453024A1 filed by vanden Bosch describes a vaccine prepared from a 42 Kd protein. This vaccine has 103/105 Kd hemolysin and 120 Kd cytotoxin and 42 Kd OMP. The protein described by this application is heat modifiable (30xc2x0-100xc2x0 C. in buffer for 10 minutes) and sarcosyl insoluble and thus this protein is different from that of the present invention.
There is a need for an effective and reliable protein vaccine which does not require other APP cell components.
It is therefore an object of the present invention to provide a novel protein vaccine which provides immunity to homologous and heterologous serotypes of a bacterium. Further, it is an object of the present invention to provide methods for producing the vaccine which are relatively easy to perform, safe and reliable. These and other objects will become increasingly apparent by reference to the following description and the drawings.